We have used PCR to simultaneously detect multiple viruses in a single PCR assay. Briefly, co-amplification was performed with primers derived from the gag region of HIV-1 (SK 38/39) and the LTR region of HIV-2 (SK 89/90), HIV-1 and HCV (hepatitis C virus) and HIV-1 and HBV. Primers were standardized for optimal sensitivity in the co-amplification reaction. Amplification was performed with primer pairs specific for a set of 2 of these viruses (HIV-1/HIV 2, HIV-1/HCV, HIV-1/HBV) and the products analyzed by hybridization with virus specific probes. Fewer than 10 copies of each viral DNA could be detected by this method. This assay was used to detect the respective viruses in co-infected clinical samples. We are currently optimizing co-amplify HIV-1/2, HBV, HCV and HTLV-I/II sequences in a single PCR assay using plasmid titration. Abstracts describing this work were presented at the VII Int. Conf. on AIDS. Florence. A manuscript has been submitted to the Journal of Virological Methods.